In eukaryotes, gene regulation is largely controlled at the transcriptional level by promoter-specific activator proteins (activators) whose DNA binding sites are typically present upstream of the core promoter of genes transcribed by RNA polymerase II (class II genes). Activators must work, directly or indirectly, through a set of general transcription factors (GTFs) that are required for transcription of all class II genes. The mechanisms by which activators stimulate transcription is the subject of this application. A variety of studies have shown that activators function at least in part by stimulating pre- initiation complex (PIC) assembly. During the past funding period we have shown how the chromatin immunoprecipitation (CHIP) assay can be used to study PIC assembly in living cells. We will continue to use this assay to study activator-mediated PIC assembly in yeast and higher eukaryotes. We have also made significant progress in our understanding of the role of TBP- Associated-Factors (TAFIIS). We will continue and extend our systematic investigation of how individual TAFIIS mediate transcriptional activation and cell cycle progression. Previous studies from our laboratory and others have implicated the GTF TFIIB in the mechanism by which some activators function. Experiments are proposed to continue to study the role of TFIIB in transcription actiation in vivo. Finally, our experience with the CHIP assay has suggested an approach for developing a general method for identifying the gene targets of activators. Such a method would be extremely useful in many areas of biology and medicine.